The long range goal of this research project is to understand the detailed structure/function relationships of the receptors for insulin and the insulin-like growth factors (IGFs), particularly with regard to their biological signaling mechanisms. Progress over the last five years supported by this grant on a number of important issues has resulted in 46 publications. Major accomplishments have included elucidation of insulin receptor and IGF-I receptor tyrosine kinase regulation by tyrosine phosphorylation in vitro and in vivo, and isolation and sequencing of cDNA clones encoding that rat IGF-II/Man-6-P receptor. Our proposed studies will focus on three critically important questions about the type I and type II insulin and IGF receptors: 1.) What is the biochemical nature and role of serine phosphorylation of insulin receptor in regulating its function (Specific Aim l)? An insulin-sensitive serine kinase activity which copurifies with our affinity-purified insulin receptor will be purified and evaluated. We shall purify this serine kinase and test whether it is activated by the insulin receptor or whether the receptor becomes better substrate upon binding insulin. 2.) Does tyrosine phosphorylation initiated by the insulin receptor kinase activate a novel membrane-bound insulin-activated serine kinase activity we recently discovered (Specific Aim 2)? We shall attempt to purify this serine kinase prepare antibodies, and assess directly whether it is tyrosine phosphorylated and activated in intact cells or in vitro. The latter experiment will involve direct phosphorylation reactions with purified serine kinase and insulin receptor kinase (and other tyrosine kinases). 3.) What is the signalling role, if any, of the IGF-II/Man-6-P receptor (specific Aims 3 and 4? This controversial issue will be addressed directly with the use of specific polyclonal antibodies against this receptor to assess whether it mediates bio-effects of IGF-II. Glycogen synthesis and calcium influx will be monitored in response to IGF-II monitor (with or without blocking antibodies) in receptor-minus mutant cell lines before and after transfection and expression of full length IGF-II/Man-6-P receptor cDNA. Frog oocytes (normally devoid of this receptor) will be microinjected with receptor RNA and possible biological effects of IGF-II monitored. In vitro mutagenesis of the receptor cDNA will also be performed in both exofacial and cytoplasmic domains to clarify roles of specific receptor structural region in ligand binding and biological signalling, if such signalling mechanisms exist.